JP2689140B2 - Novel β-hydroxysteroid oxidase and its preparation method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel β-hydroxysteroid oxidase and a method for preparing the same, which enzyme is used for steroid conversion and cholesterol quantification. It

Technical background Steroids are a general term for compounds having a steroid nucleus, and contain many biologically important substances such as bile acids, sex hormones, corticosteroids, and cholesterol. Great demand for the body.

At present, plant sterols extracted from cholesterol and soybean fat extracted from wool fat are used as starting materials for steroid synthesis. In recent years, in the synthesis of steroids, a conversion using microorganisms has been developed by Arima et al. [K. Arima et al., “Agricultural End Biological Chemistry” (Agric.Biol.C).
hem.) 33: 1636, (1969)], this method is industrially adopted.

This reaction converts cholesterol into 3β-hydroxy steroid oxidase.
The reaction of converting into cholest-4-en-3-one by the action of is started. The reaction is represented as follows:

Most steroid reactions by microorganisms are oxidation-reduction reactions, and since it is necessary to supply the coenzymes related to this to the reaction system, there is no example in which an enzyme alone is industrially used, and microbial cells are used as the enzyme system. It has been. Therefore, almost no attempts have been made to isolate the enzyme involved in steroid conversion and analyze the reaction mechanism in detail.

On the other hand, in fields such as clinical examination and biochemical analysis, cholesterol has been quantified and used for diagnosis of hyperlipidemia and arteriosclerosis. Cholesterol oxidase is used as the method for enzymatically measuring cholesterol. However, this enzyme was extracted from the enzyme produced in the inside or outside of the microorganisms of the microorganisms belonging to the genus Arthrobacter, Brevibacterium, Nocardia, Microbacterium, Pseudomonas and Streptomyces. The crude enzyme was used as is. However, there are no examples of clarifying the enzymatic properties and using the purified enzyme. A method for culturing Rhodococcus equi No. 23 strain to obtain cholesterol oxidase is disclosed in JP-A-61-247381. However, in this method, a purified enzyme has not been obtained. It has not been clarified whether the reaction system requires a coenzyme system.

DISCLOSURE OF THE INVENTION PROBLEM TO BE SOLVED BY THE INVENTION As described above, there has been no case in which a purified enzyme has been used in the conversion of steroids so far, and it is industrially usable and requires the supply of a coenzyme. Not 3-β-
Hydroxysteroid oxidase has not been obtained. Furthermore, there is no example of using purified cholesterol oxidase in the measurement of cholesterol, and the substrate specificity of the enzyme used is wide, and there is a problem in the reliability of the measurement.

INDUSTRIAL APPLICABILITY The present invention can be used industrially as well as for clinical examinations.
It is an object of the present invention to provide a novel 3-β-hydroxysteroid oxidase produced by a strain belonging to purified Rhodococcus equi that does not require the supply of a coenzyme system.

 Hereinafter, the present invention will be described in detail.

Means for Solving the Problems In order to solve the above problems, the present inventors have conducted research on an enzyme produced by a steroid-degrading microorganism, and as a result, Rhodococcus equi (Rhodococ) isolated from soil has been investigated.
cus equi) MIL 1037 strain was found to produce a novel 3-β-hydroxysteroid oxidase outside the bacterial cell, and the present invention was completed.

The strain Rhodococcus equi MIL 1037 strain used in the present invention (hereinafter referred to as 1037 strain) was isolated by the present inventors,
Micromachine research bacterium deposit No. 1017 at Institute of Microbial Science and Technology, AIST
Deposited as No. 9.

To obtain the desired 3-β-hydroxysteroid oxidase using the 1037 strain, the strain may be inoculated in a liquid medium, cultured according to a conventional method, and collected from the culture solution. It is also possible to induce a enzyme production by adding a steroid compound such as cholesterol to the medium.

The medium used for culturing may be any as long as it contains a nutrient source that can be used by strain 1037, but particularly preferable medium is a nutrient medium, a medium generally known as modified nutrient medium. Can be mentioned. A medium supplemented with 2% agar can be used as a slant for storage of this strain. The composition of the modified nutrient medium, for example, corn stapler 1.
Containing 0% by weight, beef extract 0.5% by weight, glucose 0.5% by weight and potassium phosphate 0.2% by weight (pH 7.0
Adjustment).

Examples of the culturing method include static culturing, shaking culturing, and jar fermenter culturing, and the jar fermenter is preferred for large-scale culturing. The culture conditions are
Although it varies depending on the medium and the culture vessel, it is advisable to carry out the culture at a culture temperature of 20 ° C to 35 ° C, preferably 25 ° C to 30 ° C for 24 hours to 96 hours, preferably 24 hours to 48 hours.

After completion of the culture, the bacterial cells and the culture supernatant are separated according to a conventional method. For example, a method such as centrifugation or filtration can be exemplified. From the culture supernatant from which the bacterial cells have been removed, 3-β-hydroxysteroid oxidase is separated and collected by a well-known purification means. For example, cells are removed from the culture by centrifugation, and ammonium sulfate is added to the supernatant for salting out. Then, the protein fractions precipitated by salting out were collected by centrifugation, and 10 mM sodium phosphate buffer solution (pH 7.0) was used.
And then dialyzed in the same buffer to obtain a crude enzyme solution. This crude enzyme solution can also be used as a desired enzyme for steroid conversion and as a diagnostic enzyme.

This crude enzyme solution was further added to 10 mM Tris-HCl buffer (pH
It can be fractionated and purified by dialysis with 8.0) and then sequentially passing through DEAE-cellulose column chromatography, CM-cellulose column chromatography, and hydroxyapatite column chromatography. further,
Gel filtration using Sephadex G-75 can be performed to obtain the final purified enzyme. 3-β purified in this way
-Hydroxysteroid oxidase shows a single spot by SDS electrophoresis.

The activity of the enzyme thus obtained is measured as follows.

(1) Measurement of enzyme activity 10 mM sodium phosphate buffer (pH 7.
0) 1 ml was put in a reaction tank (1 ml volume), saturated oxygen was dissolved, and then cholesterol (1 μmol / 20 μl ethanol solution)
Is added and the reaction is carried out at 30 ° C., and the consumption of dissolved oxygen after 1 minute is determined. In addition, a dissolved oxygen meter for biochemistry, Oxygraph 8 (manufactured by Central Kagaku) was used to measure the dissolved oxygen, and the enzyme activity was 1 unit (U ), The specific activity is 1 mg protein
Expressed as enzyme units (U / mg).

(2) Protein quantification method Protein quantification is performed by the Lowry method using bovine serum albumin (manufactured by Sigma) as a standard.

The specific activity of the purified enzyme thus measured is 20 U / mg protein or more.

The 3-β-hydroxysteroid oxidase purified as described above has the following enzymatic chemical properties.

Action The enzyme activity was measured using various steroid compounds as substrates. When the oxygen consumption of each substrate was measured with the dissolved oxygen consumption of cholesterol as the substrate being 100, the results shown in Table 1 were obtained. The enzyme shows activity only on a substrate having a 3β hydroxyl group.

Molecular weight The molecular weight was 48,000 ± 2,000 when measured by SDS polyacrylamide gel disc electrophoresis.

Isoelectric point As a result of sucrose density gradient isoelectric focusing, the isoelectric point is pH 9.2.
It was ± 0.1.

Optimum pH The optimum pH was 7.5. (See FIG. 1) pH stability It was stable at pH 7.0 to pH 9.0 in each treatment at 4 ° C. for 1 hour and 4 ° C. for 24 hours. (See Fig. 2) Optimum temperature The working temperature ranges from 5 ℃ to 55 ℃, and the optimum temperature is 45 ℃.
It was. (See Fig. 3) Heat stability When treated at pH 7.0, heating at 50 ° C for 1 hour shows 30% residual activity, and heating at 60 ° C for 5 minutes shows 15% residual activity. It was (See FIG. 4) Metal binding EDTA, o-phenanthroline, α-α′-dipyridyl-
The activity is not inhibited by chelating agents such as 8-quinolinol, potassium cyanide and sodium nitrate. From this, it was confirmed that there was no metal ion in the enzyme.

Effects of metal salts Metal salts CaCl ₂ , MgCl ₂ , FeCl ₂ , FeSO ₄ , CuSO ₄ , Na ₂ Mo
Add O ₄ , CoCl ₂ , NiCl ₂ , MnCl ₂ , AgNO _{3 and} HgCl ₂ to 10 ^-2 , 1
The effects were examined by adding 1 ml at the time of measuring the enzyme activity, which was adjusted to the concentration of 0 ^-3 and 10 ^-4 M / ml respectively. The results are shown in Table 2, and AgNO ₃ and HgCl ₂ strongly suppressed the enzyme activity. or,
This inhibition was restored by the addition of 10 mM glutathione and dithiothreitol.

Km, Vmax value Km value when using cholesterol as a substrate is 3.27 × 10 ^-4 M,
The Vmax at the enzyme concentration of 5 μg / ml is 1.33 × 10 ⁻⁴ M.

Absorption spectrum Hitachi 100-60 type double beam spectrophotometer (manufactured by Hitachi Ltd.)
In the absorption spectrum using, it had a maximum absorption at 280 nm and had no other absorption. (See FIG. 5) Since this enzyme has no FAD absorption, it is clear that it has different properties from FDA-dependent 3β-hydroxysteroid oxidase.

Hereinafter, the present invention will be described more specifically with reference to examples.

Example 1 Preparation of culture solution Rhodococcus equi MIL 1037 strain (Microtech Lab.
One platinum loop from the storage slant was inoculated into a slant medium for seeds, and static culture was carried out at 30 ° C. for 24 hours to obtain a slant for seeds. From this slant for seed, 3 platinum loops were added to a nutrient medium (100 ml / 500 ml
(Triangular Kolben) and subjected to rotary shaking culture (200 rpm) at 27 ° C. for 24 hours, which was used as preculture. Furthermore, 3 ml from the preculture was inoculated into 100 ml of modified nutrient medium,
Similarly, the culture was cultivated with rotation shaking at 27 ° C. to obtain a culture solution.

Modified Nutrient Medium Composition: Corn stapler 1.0wt% Beef extract 0.5wt% Glucose 0.5wt% K ₂ HPO ₄ 0.2wt% pH 7.2 Purification of enzyme Collect the culture solution obtained as above 15 at 12,000G
After centrifugation for 10 minutes, 10 l of culture supernatant was obtained. Ammonium sulfate was added to this supernatant for saturation fractionation. Saturated fractionation was performed at 20, 30, 40, 50, 60, 70 and 80 wt% ammonium sulfate, respectively. Protein precipitation at 12,000 G for 15 minutes at 4 ° C
Were collected by centrifugation at 10, and suspended in 10 mM sodium phosphate buffer (pH 7.0). The suspension should be 5 l with the same buffer, 5 l.
After dialysis once for 48 hours, enzyme activity and protein amount were measured. The 50% and 60% highly active saturated fractions were combined to make a crude enzyme solution, and 5 mM of 10 mM Tris-HCl buffer (pH 8.0) was added.
Dialysis was performed 5 times for 48 hours.

 This enzyme solution was used as a crude enzyme solution.

550 ml of crude enzyme solution was added to 10 mM Tris-HCl buffer (pH 8.0)
DEAE-cellulose column (inner diameter 2.6 cm ×
(Length 60 cm) and eluted with the same buffer. Total activity 2U / 1
Fractions over 0 ml were combined, concentrated in an evaporator at 30 ° C, and 4 times with 5 l of 10 mM sodium phosphate buffer (pH 7.0) 4 times.
After dialysis for 8 hours, 150 ml of enzyme solution was obtained. 10 mM of this enzyme solution
It was applied to a CM-cellulose column (inner diameter 2.6 cm x length 60 cm) equilibrated with sodium phosphate buffer (pH 7.0). The column was flushed with 10 mM of the same buffer, washed, and then eluted with 25 mM of the same buffer (pH 7.0) at a flow rate of 1.0 ml / min.
The active fractions were collected, concentrated in an evaporator at 30 ° C., and dialyzed against 3 l of 10 mM sodium phosphate buffer (pH 7.0) four times for 48 hours to obtain 50 ml of enzyme solution. 10 mM of this 50 ml enzyme solution
It was applied to a hydroxyapatite column (inner diameter 0.5 cm × length 15 cm) equilibrated with sodium phosphate buffer (pH 7.0). 50 mM of the same buffer (pH
7.0) and then eluted with 100 mM of the same buffer (pH 7.0). The active fractions were collected, concentrated at 30 ° C using an evaporator, and then concentrated with 3 l of 10 mM sodium phosphate buffer (pH 7.0).
It was dialyzed once for 48 hours and 5 ml of the enzyme solution was recovered.

5 ml of enzyme solution obtained by hydroxyapatite column
Was swelled and equilibrated with a 10 mM sodium phosphate buffer (pH 7.0) to a Sephadex G-25 column (inner diameter 2.6 cm x length 60 c).
m) and eluted with the same buffer. The active fractions (see FIG. 6) were collected and concentrated to 10 mM sodium phosphate buffer (pH 7.
0) It dialyzed 4 times with 3l for 48 hours. Then, freeze-drying was performed to obtain 22 mg of purified enzyme. The specific activity of this enzyme is 21.4
It was 0 U / mg.

The purification degree and recovery rate of each purification step are as shown in Table 3.

Identification of Enzyme Protein 50 μg of the enzyme protein was subjected to gel desk electrophoresis using polyacrylamide. It was performed at 4 mA for 2 hours using pH 9.4, 7.5% T gel, and after electrophoresis, it was stained with 0.25% Coomassie Brilliant Blue R-250. (See FIG. 7) As shown in the figure, a single band was shown, and it was confirmed that the product was uniformly purified.

Effects of the Invention According to the present invention, a novel 3β-hydroxysteroid oxidase can be obtained, and the present enzyme is a coenzyme.
In the absence of FAD, it has an effect of converting cholesterol into cholest-4-en-3-one, which is a starting material for steroid synthesis, and thus can be effectively used for conversion of steroid and quantification of cholesterol.

[Brief description of the drawings]

FIG. 1 shows the optimum pH of the enzyme according to the present invention, FIG. 2 shows the pH stability of the enzyme, FIG. 3 shows the optimum temperature of the enzyme, and FIG. 4 shows the optimum temperature of the enzyme. It shows thermostability, and FIG. 5 shows the absorption spectrum of this enzyme. Further, FIG. 6 shows the active fraction of this enzyme eluted from the Sephadex G column, and FIG. 7 shows the stained band of this enzyme protein by electrophoresis.

Claims (4)

(57) [Claims] 1. A β-hydroxysteroid oxidase having the following enzymatic properties: Action: A 3-oxosteroid is produced by specifically acting on a steroid having a 3β-hydroxyl group. Molecular weight: 48,000 ± 2,000 Isoelectric point: pH 9.2 ± 0.1 Optimal pH: 7.5 pH stability: Stable in the pH range of 7.0 to 9.0 when treated at 4 ° C for 24 hours. Optimum temperature: 45 ° C Thermal stability: 90% at 40 ° C when treated at pH 7.0 for 1 hour
The above activity is retained. Metal bond: No metal ion is present in the enzyme molecule. Effect of metal salt: silver nitrate (AgNO _3), mercuric chloride (HGC
The enzyme activity is inhibited by l ₂ ) and the activity is restored by the addition of glutathione and dithiothreitol. Km and Vmax values: Km value when cholesterol is used as a substrate
Vmax value of 1.33 × 10 at 3.27 × 10 ^-4 M and enzyme concentration of 5 μg / ml
^-4 M. Absorption spectrum: No absorption other than protein is observed in the ultraviolet and visible absorption spectra. 2. Rhodococcus equ
The β-hydroxysteroid oxidase according to claim 1, which is produced by i ). 3. A method for culturing Rhodococcus equi, and separating and collecting β-hydroxysteroid oxidase having the enzymatic property of claim 1 from the resulting culture solution. Β described in item (1)
-Method for preparing hydroxysteroid oxidase. 4. The method according to claim 3, wherein the Rhodococcus equi is Rhodococcus equi MIL1037 strain (Ministry of Industrial Science and Technology No. 10179 ).